Anti-Neprilysin Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight band should I expect for Neprilysin on Western blot?

Neprilysin migrates as a single band at approximately 90-100 kDa on reducing SDS-PAGE, reflecting the full-length glycosylated protein. The predicted molecular weight from the amino acid sequence is around 86 kDa, but N-glycosylation at multiple sites increases the apparent mass. If you see multiple bands, consider that proteolytic cleavage can generate smaller fragments, particularly in samples with active proteases. Including protease inhibitors during lysis is critical. The type II membrane topology means the bulk of the protein is extracellular, so expect robust signal in membrane fractions and lower abundance in cytosolic preparations.

What starting dilution should I use for this Neprilysin antibody in Western blot?

We recommend starting at 1:1000 dilution for Western blot based on validation data with human samples. This dilution typically provides clean signal with low background when using standard chemiluminescent detection. If your lysate has low Neprilysin expression or you are working with tissues other than kidney or brain, you may need to increase concentration to 1:500. Conversely, kidney cortex and certain tumor lines express high levels and may permit dilution to 1:2000. Always include a titration series with your first sample type to establish optimal signal-to-noise for your specific application and detection system.

Is this Neprilysin antibody likely to work in mouse or rat samples?

This antibody is validated for human Neprilysin. Mouse and rat Neprilysin share approximately 85-88 percent sequence identity with human, so cross-reactivity is possible but not guaranteed. If you plan to use mouse or rat samples, we recommend testing a dilution series starting at 1:500 with a positive control tissue such as kidney cortex, which expresses high endogenous Neprilysin across species. Include a known mouse or rat Neprilysin-positive lysate if available. Cross-reactivity should be confirmed empirically in your hands; we cannot provide validated dilutions for species beyond those explicitly tested.

What are good positive and negative control tissues for Neprilysin expression?

Kidney cortex is the gold-standard positive control, showing robust Neprilysin expression in proximal tubule brush border membranes. Brain tissue, particularly striatum and cortex, also expresses significant levels in neuronal membranes. For cell lines, certain leukemias and lymphomas express CD10/Neprilysin strongly. Negative controls include most hematopoietic cells outside the lymphoid lineage and hepatocytes, which have minimal to undetectable expression. When troubleshooting, compare signal intensity between kidney and a low-expression tissue on the same blot. This internal comparison confirms antibody specificity and helps optimize dilution for less abundant samples.

Does Neprilysin have splice isoforms I need to consider when interpreting Western blots?

Neprilysin is predominantly expressed as a single full-length isoform encoded by the MME gene. Alternative splicing has been reported in the literature but is rare and not well-characterized functionally. You should expect one major band around 90-100 kDa in most samples. Soluble forms of Neprilysin generated by ectodomain shedding have been described and may appear as slightly smaller bands if present in serum or conditioned media. If you observe multiple bands in cell lysates, suspect post-translational modifications such as differential glycosylation or proteolytic processing rather than splice variants. Review your sample preparation to ensure protease inhibitors are included.

Can I use this antibody for immunohistochemistry or immunofluorescence on Neprilysin?

Triple Point Biologics antibodies are validated for Western blot and immunohistochemistry as standard applications. This rabbit polyclonal should be suitable for IHC and immunofluorescence on fixed tissue sections or cultured cells, though optimal conditions must be determined empirically. For IHC, start with a 1:100 to 1:200 dilution and antigen retrieval in citrate buffer. Neprilysin localizes to the plasma membrane, so expect rim staining in positive cells. Kidney sections provide excellent positive controls with strong staining in proximal tubule brush borders. Always include secondary-only controls to assess non-specific binding, particularly in tissues with high endogenous immunoglobulin.

How should I prepare cell lysates to preserve Neprilysin for Western blot detection?

Neprilysin is a membrane-bound metallopeptidase, so use lysis buffers containing non-ionic detergents such as 1 percent Triton X-100 or NP-40 to solubilize membranes efficiently. Include protease inhibitors immediately upon lysis; Neprilysin itself is a protease and sample degradation is common without inhibition. EDTA-containing inhibitor cocktails are acceptable and will not interfere with antibody recognition. Avoid repeated freeze-thaw cycles, which can promote aggregation of membrane proteins. For subcellular fractionation studies, Neprilysin partitions primarily with membrane fractions. Load 20-40 micrograms total protein per lane as a starting point for detection in moderate-expression samples.

What is the recommended storage condition and shelf life for this Neprilysin antibody?

Store the antibody at -20 degrees Celsius in single-use aliquots to avoid repeated freeze-thaw cycles, which can reduce titer and increase aggregation. The antibody is supplied at 100 micrograms per vial in a stabilization buffer. Once thawed, aliquots remain stable for at least six months at -20 degrees Celsius and can be kept at 4 degrees Celsius for up to one month for frequent use. Do not store diluted antibody; prepare working dilutions fresh in blocking buffer on the day of use. If you observe increased background or loss of signal over time, prepare a fresh aliquot. Avoid contamination by using sterile technique when removing aliquots from stock vials.